PROJECT SUMMARY Inflammation in the brain induced by glial activation and disruption of glutamate neurotransmission have been implicated in the pathogenesis and worsening of symptoms in Rett syndrome (RTT), a debilitating, developmental disorder that is associated with seizures, intellectual disability, motor and autonomic dysfunction, and non-purposeful hand movements. Activated microglial cells and astrocytes mediate the neuroinflammatory response that leads to oxidative injury and increased glutamate toxicity. Therapies targeted to these ?activated? microglia for diminishing inflammation, oxidative injury and excess glutamate production could have an impact in RTT by arresting the injury and promoting repair and regeneration. In addition to over production of glutamate by these mechanisms, glutamate receptor expression is altered in RTT and mouse models of Mecp2 insufficiency that may contribute to synaptic dysfunction in RTT. Building on our positive preliminary results, we propose an innovative, nanotherapeutic approach to attenuate/arrest the injury in RTT. Using dendrimers that intrinsically target activated microglia and astrocytes upon systemic administration, we will deliver specific drugs to down regulate oxidative stress, glutamate production and inflammation. We have previously shown that dendrimers can target activated glia and deliver drugs to produce significant efficacies, in models of cerebral palsy and neonatal stroke. Our preliminary results in a mouse model of RTT indicate that dendrimer nanodevices localize in microglia and astrocytes of hemizygous male (Mecp2-null) mice but not in wild-type (WT) mice. Moreover, dendrimer-conjugated N-acetyl cysteine (D-NAC) delivered systemically weekly from 3 weeks of age, when Mecp2-null mice become symptomatic, results in significant improvement in neurobehavioral scores at 6-7 weeks of age. We hypothesize that targeted delivery of an anti- inflammatory/anti-oxidant along with an anti-glutaminase agent to activated microglia and astrocytes in Mecp2- null and Mecp2-heterozygous (HET) mice will lead to decreased oxidative injury and glutamate toxicity resulting in improved neurobehavioral outcomes and symptom free survival. This will be evaluated using dendrimer conjugated to N-acetyl cysteine (D-NAC; anti-inflammatory/anti-oxidant) and dendrimer conjugated to a novel analogue of BPTES (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide; D-JHU 29; glutaminase inhibitor). Our Specific Aims are: 1) to evaluate the efficacy of monotherapy with D-NAC or D- JHU29 in Mecp2-null and HET mice, and 2) to determine whether combination therapy with D-NAC and D-JHU will be more effective than monotherapy in improving survival and long-term neurologic outcomes. Effects on survival, weight, behavior, vision, respiration, glutamate and glutathione levels and oxidative stress, and brain volume will be evaluated. These initial proof-of-concept studies will not only provide a greater understanding of the role of activated glia in RTT, but will also form the basis for future studies enabling clinical translation.